Methods and systems for phylogenetic analysis

Abstract

The present invention discloses methods and systems for designing and using organism-specific and/or operational taxon unit (OTU)-specific probes. The methods and systems allow for detecting, identifying and quantitating a plurality of biomolecules or microrganisms in a sample based on the hybridization or binding of target molecules in the sample with the probes. Some embodiments provide methods of selecting an oligonucleotide probe specific for a node on a clustering tree. Other embodiments provide methods of selecting organism-specific or OTU-specific oligonucleotide probes for use in accurately detecting a plurality of organisms in a sample with high confidence. Some embodiments provide methods and systems to detect the presence of a rare OTU in a sample.

1 .- 110 . (canceled) 
     
     
         111 . A system capable of detecting one or more first nucleic acid sequences in a population of nucleic acid sequences in a single assay, wherein the one or more first nucleic acid sequences comprise less than 0.01 percent of the total nucleic acids in the population of nucleic acid sequences and wherein the detection is with a confidence level greater than 95% and a sensitivity level greater than 95%, and wherein the one or more first nucleic acid sequences are at least 95% homologous to all of the nucleic acid sequences in the population of nucleic acid sequences. 
     
     
         112 . The system of  claim 111  wherein said system is configured for high-throughput sequence analysis of gut microbial communities. 
     
     
         113 . The system of  claim 111  wherein said system detects presence, absence, relative abundance, and/or quantity of more than 10,000 different Operational Taxon Units (OTUs) of a single domain in a single assay with confidence greater than 95%. 
     
     
         114 . The system of  claim 111 , wherein one or more of said first nucleic acid sequences are 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, cox1 gene, nif13 gene, RNA molecules derived therefrom, or a combination thereof. 
     
     
         115 . A system for determining the presence, absence, relative abundance, and/or quantity of a plurality different OTUs in a single assay, said system comprising a plurality of polynucleotide interrogation probes, a plurality of polynucleotide positive control probes, and a plurality of polynucleotide negative control probes. 
     
     
         116 . A method comprising: measuring an increase of a particular microorganism's percentage of gut microbiome in an individual using the system of  claim 111 . 
     
     
         117 . A method for determining the probability of the presence or quantity of a unique sequence or microorganism in a sample comprising: a) contacting said sample with a plurality of different probes; b) determining hybridization signal strength for sample sequences to each of said probes; c) removing or attenuating from analysis an OTU from the possible list based on hybridization signal strength data, thereby increasing the confidence in the remaining hybridization signal strength data. 
     
     
         118 . The method of  claim 117  wherein the removing or attenuating is performed by penalizing the likelihood that an OTU is present in the sample based on potential cross hybridization of probes from said OTU with sequences from other OTUs. 
     
     
         119 . The method of  claim 117  wherein the method comprises determining GC content of each probe in the system and using a d score that compares each probe intensity to a positive control probe intensity and negative probe intensity to determine quantity of said probes. 
     
     
         120 . The method of  claim 118 , wherein the greater the potential for cross hybridization, the greater the penalization. 
     
     
         121 . The method of  claim 120 , wherein the penalization based on cross hybridization is performed at each level of a phylogenic tree starting with the lowest level. 
     
     
         122 . A method for identifying a microbiome signature indicative of a condition comprising: a) comparing the presence and optionally abundance of at least one of more different OTUs in a control sample without said condition and a reference sample with said condition using the system of  claim 111 ; and b) identifying one or more OTUs that associate with said condition. 
     
     
         123 . The method of  claim 122  wherein said reference and control samples are obtained from the gut, respiratory system, oral cavity, sinuses, nares, urogenital tract, skin, feces, udders, auditory canal, blood, sputum, urine or a combination thereof. 
     
     
         124 . The method of  claim 122  wherein said condition is one of the human gut. 
     
     
         125 . The method of  claim 122 , wherein said condition is Crohn's Disease, irritable bowel syndrome, stomach ulcers, colitis, neonatal necrotizing enterocolitis, or gastroesophageal reflux disease. 
     
     
         126 . The method of  claim 122 , wherein the condition is cystic fibrosis, chronic obstructive pulmonary disease, rhinitis, atopy, asthma, acne, food allergy, obesity, or periodontal disease. 
     
     
         127 . The method of  claim 122 , wherein said condition is any disease or disorder caused by, aggravated by or related to the presence, absence or population change of a microorganism. 
     
     
         128 . The method of  claim 122 , wherein an increase in the similarity in the presence and optionally abundance of said OTUs in said reference sample with respect to said control sample is indicative of remediation of said condition. 
     
     
         129 . The method of  claim 122 , wherein changes in the degree of similarity in the presence and optionally abundance of said OTUs in said reference sample with respect to said control sample are provided as a measure of remediation of said condition. 
     
     
         130 . The method of  claim 122  wherein said microbiome signature consists of up to 200 different OTUs that associate with said condition.